ИСТИНА |
Войти в систему Регистрация |
|
ИСТИНА ЦЭМИ РАН |
||
The authentic translation initiation mechanism of unspliced HIV-1 mRNA remains disputable. While this mRNA is naturally capped and there is little doubt that it can be translated via the cap-dependent mechanism, numerous reports stated that it also may initiate translation in a cap-independent fashion. The first paper to announce existence of an Internal Ribosome Entry Site in the leader of HIV-1 unspliced mRNA was published about a decade ago. Since then this issue has remained controversial, being confirmed in some reports but not validated in others. The consensus of opinions is that HIV-1 5’UTR IRES functions when cap-dependent translation is inhibited; more specifically it was suggested to be active in G2/M-phase. However, the question of which mechanism prevails, if any, is still unclear. Here we have addressed the potential of the unspliced HIV-1 mRNA 5‘UTR to function as an IRES using our previously reported stringent criteria, which ground on direct comparison of monocistronic and bicistronic mRNAs translation efficiency. Such an approach enables one to address contribution of both mechanisms to the overall level of capped monocistronic mRNA translation. Importantly, we have committed to transfections of RNA, rather then DNA, given the latter approach’s strong bias towards producing artifacts. We were able to show that under all conditions tested the primary mechanism used by 5’-UTR of HIV-1 unspliced mRNA is 5’-end dependent scanning. Absence of an IRES and relatively high cap-dependence of RNA with this leader were confirmed both in vitro (translation in cell lysates) and in vivo (RNA-transfection of several cell lines, including those derived from T-cells). Hypothesis of cell cycle dependent IRES, which is active in G2/M phase when cap-dependent translation is suppressed, has also been disproved with transfection of synchronized cells or under conditions when cap-dependent translation was directly inhibited.